Localization of99mTc in bone by means of autoradiography

The uptake of two different preparations of^99mTechnetium-methylene diphosphonate in fetal rat calvaria is compared. The localization of^99mTc after administration of^99mTc(Sn)-MDP and^99mTc-MDP showed equal distribution in autoradiography.     

The Effect of IFN-gamma, Alum and Complete Freund Adjuvant on TNP-KLH Induced Ig.G(1), IgE and IgG(2a) Responses in Mice

Adjuvants are considered to play an important role in directing the isotype and amount of antibodies produced upon immunization by conducting the development of either Th-1 or Th-2 cells upon T-cell stimulation. This is based on the different cytokine production patterns that were observed after in vitro resttmulation of T cells isolated from mice immunized with antigen either adsorbed on alum or emulsified in complete Freund adjuvant (CFA). However, other studies suggest that primarily the type of antigen determines which isotypes are produced and to what extent. In these studies, however, IgE was not determined. Therefore, this study examined whether alum and CFA influenced the amount and/or ratio of IgG(1), IgE and IgG(2a) produced after TNP-KLH immunization. Similar levels of IgG(1), IgE and IgG(2a) antibodies were found upon immunization with TNP-KLH either adsorbed on alum or emulsified in CFA. Moreover, administration of IFN-gamma in combination with TNP-KLH adsorbed on alum did not increase the amount of IgG(2a) produced. IFN-gamma treatment resulted in an increased IL-6 and decreased IFN-gamma production by spleen cells upon Con A stimulation, whereas it did not change the IL-4 production in similar conditions. The presented results suggest that upon immunization with TNP-KLH high IL-4 levels are produced, resulting in an antibody response that is dominated by IgG(1), independent of the adjuvant employed. The IL-4 inducing property of TNP-KLH is substantiated by the finding that repeated immunization of mice with TNP-KI, without adjuvant, increases the serum total IgE level. The presented data suggest that the carrier part of TNP-KLH preferentially results in Th-2 cell activity after which the adjuvant merely enhances the antibody responses generated.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Comparison of Pathogen DNA Isolation Methods from Large Volumes of Whole Blood to Improve Molecular Diagnosis of Bloodstream Infections

For patients suffering from bloodstream infections (BSI) molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1–5 ml), the novel non-enzymatic procedure (Polaris, 1–5 ml), and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl). These methods were evaluated by processing blood spiked with 0–1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1–10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct) values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50–67%) and EasyMAG (58–79%). For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70–75% (MolYsis 17–50% and TTE-EasyMAG 20–36%). The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction) vs. 2 hours (MolYsis)). In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.     

A second gene at the tomato Cf-4 locus confers resistance to cladosporium fulvum through recognition of a novel avirulence determinant

The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homologous genes (Hcr9-4s) at this locus. Cf-4 was identified by molecular analysis of rare Cf-4/Cf-9 disease-sensitive recombinants and by complementation analysis. The analysis did not exclude the possibility that an additional gene(s) located distal to Cf-4 may also confer resistance to C. fulvum. We demonstrate that a number of Dissociation-tagged Cf-4 mutants, identified on the basis of their insensitivity to Avr4, are still resistant to infection by C. fulvum race 5. Molecular analysis of 16 Cf-4 mutants, most of which have small chromosomal deletions in this region, suggested the additional resistance specificity is encoded by Hcr9-4E. Hcr9-4E recognizes a novel C. fulvum avirulence determinant that we have designated Avr4E.     

Detection of Helicobacter species DNA by quantitative PCR in the gastrointestinal tract of healthy individuals and of patients with inflammatory bowel disease

In many animal species different intestinal Helicobacter species have been described and a few species are associated with intestinal infection. In humans, the only member of the Helicobacter family which is well described in literature is Helicobacter pylori. No other Helicobacter-associated diseases have definitely been shown in humans. We developed a sensitive quantitative PCR to investigate whether Helicobacter species DNA can be detected in the human gastrointestinal tract. We tested gastric biopsies (including biopsies from H. pylori positive persons), intestinal mucosal biopsies and fecal samples from healthy persons, and intestinal mucosal biopsies from patients with inflammatory bowel disease (IBD) for the presence of Helicobacter species. All gastric biopsies, positive for H. pylori by culture, were also positive in our newly developed PCR. No Helicobacter species were found in the mucosal biopsies from patients with IBD (n = 50) nor from healthy controls (n = 25). All fecal samples were negative. Our study suggests that Helicobacter species, other than H. pylori, are not present in the normal human gastrointestinal flora and our results do not support a role of Helicobacter species in IBD.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).